Stress-induced C-terminal lysine acetylation of p53 plays an important role in the activity of p53 as a transcription factor that regulates cell cycle arrest, senescence or apoptosis. The long-term goal of this Project is to seek mechanistic understanding of the molecular interactions that regulate the tumor suppressor p53. While multiple acetylation sites in its C-terminal tail have been reported, specific effects of individual or combined acetylation of these lysine residues on p53 activity remain elusive. Preliminary data is presented involving a structure-based functional analysis of p53 supporting the notion that acetylation-induced p53 activation in response to DNA damage is involved in co-activator recruitment and subsequent histone acetylation. This study revealed that p53 recruitment of the co-activator CBP (CREB binding protein) requires association of the conserved bromodomain of CBP with p53 at acetylated lys382: a specific molecular interaction that is essential for p53-induced transcriptional activation of the cyclin-dependent kinase inhibitor p21, involved in G1 cell cycle arrest. We hypothesize that distinct modifications of p53 C-terminal residues, including lysine acetylation and ubiquitination, as well as serine phosphorylation, have differential effects on p53 functions in cells under different conditions. A multifaceted approach is proposed to address mechanistic underpinnings of p53 transcriptional activation with the emphasis on the role of C-terminal post-translational modifications in p53 activation. The specific aims include (1) to explore the role of C-terminal modifications of p53 in co-activator recruitment using NMR structure-based biochemical analysis (2) to develop small molecule ligands to be used as probes for molecular functions of p53 using NMR-based chemical screening, and finally (3) to elucidate the role of the C-terminus of p53 in its ability to act as a transcription factor using a variety of biochemical and cell biological approaches including the establishment of an in vivo model. The proposed multidisciplinary studies range from structure-based NMR analysis and design of chemical compounds in vitro to functional analyses in cell culture, as well as the use of an in vivo mouse model. The emerging results from the proposed studies are expected to enhance our understanding of the molecular basis of C-terminal modifications in p53 function. Given the central role of p53 in cancer, these studies will have important implications for the prognosis and treatment of human tumors.